Biosurfactants are biodegradable amphiphilic molecules produced by several microbes that have excellent surface tension lowering activity and act as excellent emulsifiers, foaming and dispersing agents [Desai, J. D. and Banat, I. M. (1997) Microbiol. Mol. Biol. Rev. 61, 47-64]. These compounds are produced extracellular with potential applications in agriculture, cosmetics, pharmaceuticals, detergents, food processing, paint industry and others [Banat, I. M. (1995) Biores. Technol. 51, 1-12]. These surface-active agents are chemically diversified from low molecular weight glycolipids, lipopeptides/lipoproteins and phospholipids to high molecular weight neutral lipids, substituted fatty acids and polysaccharides [Cooper, D. G. and Zajic, J. E. (1980) Appl. Environ. Microbiol. 26, 229-256]. The microbial exopolysaccharides (EPS) with novel chemical structures offer a number of applications in food, pharmaceutical, textile, cosmetics, microbial ecology, medicine, dairy industry, biofilms, corrosion, etc. due to their rheological properties [Nicolaus, B. Kambourova, M. and Oner, E. T. (2010) Environ. Technol. 31, 1145-1158]. Some of the bacterial EPS that have found diverse range of applications are the xanthan (Xanthomonas campestris), dextran (Leuconostoc mesentroides), alginate (Pseudomonas aeruginosa and Azotobacter vinelandii), emulsan (Acinetobacter calcoaceticus), gellan (Sphingomonas paucimobilis), bacterial cellulose (Acetobacter xylinum), curdlan (Rhizobium meliloli and Agrobacterium radiobacter), succinoglycan (Alcaligenes faecalis var. myxogenes) and hyaluronic acid (Streptococcus zooepidemicus and Streptococcus equi) [Kumar, A. S., Mody, K. and Jha, B. (2007) J. Basic Microbiol. 47, 103-117]. Further, several biologically active polysaccharides such as krestin from Trametes versicolor, hetero-β-glucans from Agaricus blazei, lentinan from Lentinus edodes, and schizophyllan from Schizophyllum commune are well documented [Novak, M. and Vetvicka, V. (2008) J. Immunotoxicol. 5, 47-57]. They exhibit a diverse range of biological activities including hypoglycemic, anti-inflammatory, antitumor, anti-metastasis, hypolipidemic, immunomodulatory and antioxidant effects [Wasser, S. P. (2002) Appl. Microbiol. Biotechnol. 60, 258-274; Lee, H. H., Lee, J. S., Cho, J. Y., Kim, Y. E. and Hong, E. K. (2009) J. Microbiol. Biotechnol. 19, 566-572].
Macrophages play a key role in innate and adaptive immune system and are also involved in mounting an inflammatory response [Beutler, B. (2004). Mol. Immunol. 40, 845-859]. The activated macrophages release inflammatory mediators such as NO, TNF-α, IL-1β and IL-6 that regulate homeostasis under physiological conditions, while unregulated release was observed in several pathological conditions [Micking, J., Xie, Q. W. and Nathan, C. (1997) Annu. Rev. Immunol. 15, 323-350]. The impaired macrophage activation was observed under several pathological conditions such as septic shock, cerebral injury, myocardial ischemia, local or systemic inflammatory disorders, diabetes and other diseases. Hence, the modulation of macrophage activity is of central importance. Some microbial polysaccharides such as hetero-β-glucans extracted from fruiting bodies of Agaricus blazei exhibited immunomodulating properties [Cho, S. M., Park, J. S., Kim, K. P., Cha, D. Y., Kim, H. M. and Yoo, I. D. (1999) Korean J. Mycol. 27, 170-174]. Published reports on EPS demonstrated that they can scavenge ROS exhibiting potential antioxidant activities [Sun, C., Wang, J. W., Fang, L., Gao, X.-D. and Tan, R.-X. (2004) Life Sci. 75, 1063-1073] as well as immunostimulant activities on macrophages [Lee, H. H., Lee, J. S., Cho, J. Y., Kim, Y. E. and Hong, E. K. (2009a) J. Microbiol. Biotechnol. 19, 566-572; Lee, J. S., Min, K. M., Cho, J. Y. and Hong, E. K. (2009b) J. Microbiol. Biotechnol. 19, 951-959]. The upregulated ROS and RNS are involved in several pathological conditions and are known to cause oxidative damage to cell membrane, proteins, DNA and lipid molecules [Medzhitov, R. and Janeway, C. (2000) Immunol. Rev. 173, 89-97]. This oxidative stress induces the upregulation of transcriptional factors which in turn upregulates various proinflammatory molecules in macrophages thereby regulating various aspects of immune system [Brown, D. M., Donaldson, K., Borm, P. J., Schins, R. P., Dehnhardt, M., Gilmour, P. Jimenez, L. A. and Stone, V. (2004) Am. J. Physiol. Lung Cell Mol. Physiol. 286, L344-353].
Some Microbacterium species are reported to produce EPS. The EPS production by most of the family members of the genus Microbacterium has not been fully understood, nevertheless both homo- and hetero-types of biopolymers with glucose, mannose and fructose backbone have been reported [Bae, I. Y., Oh, I.-K., Lee, S., Yoo, S.-H. and Lee, H. G. (2008) Int. J. Biol. Macromol. 42, 10-13; Asker, M. M. S., Ahmed, Y. M. and Ramdan, M. F. (2009) Carbohydr. Polym. 77, 563-567; Godinho, A. L. and Bhosle, S. (2009) Curr. Microbiol. 58, 616-621]. Microbacterium kitamiense strain Kitami C2, isolated from the wastewater of a sugar-beet factory was reported to produce EPS [Matsuyama, H., Kawasaki, K., Yumoto, I. and Shida, O. (1999) Int. J. Syst. Bacteriol. 49, 1353-1357]. Microbacterium strain MC3B-10, isolated from the tropical intertidal rocky shore in southern Gulf of Mexico (Campeche, Mexico), produced a polymer which was not a polysaccharide but a glycoprotein with surfactant properties. This glycoprotein was rich in protein (36%) and had low levels of neutral sugars [Ortega-Morales, B. O., Santiago-Garcia, J. L., Chan-Bacab, M. J., Moppert, X., Miranda-Tello, E., Fardeau, M. L., Carrero, J. C., Bartolo-Pérez, P., Valadéz-González, A. and Guezennec, J. (2007) J. Appl. Microbiol. 102, 254-264]. Microbacterium lerregens produced an EPS exhibiting antioxidant activity with an IC50 value of 230 μg mL−1 [Asker, M. M. S., Ahmed, Y. M. and Ramdan, M. F. (2009) Carbohydr. Polym. 77, 563-567]. Microbacterium arborescencs strain AGSB, isolated from the rhizosphere of Ipomoea pescaprae, produced a mannose-based heteropolysaccharide, which had the ability to aggregate sand and improve the moisture-holding capacity [Godinho, A. L. and Bhosle, S. (2009) Curr. Microbiol. 58, 616-621]. Microbacterium sp. strain Mc1, isolated from mangrove sediment, produced a bioemulsifier which had the ability to remove cadmium and zinc from a hazardous industrial waste [Aniszewski, E., Peixoto, R. S., Mota, F. F., Leite, S. G. F. and Rosado, A. S. (2010) Brazilian J. Microbiol. 41, 235-245].
Very few strains of Brevibacillus sp. are reported to produce biosurfactants. Brevibacillus brevis HOB1 produced a lipopeptide with antibacterial and antifungal activities [Haddad, N. I. A., Wang, J. and Mu, B. (2008) J. Ind. Microbiol. Biolechnol. 35, 1597-1604], while Brevibacterium sp. strain PDM-3 produced a biosurfactant that finds application in the bioremediation of phenanthrene and polyaromatic hydrocarbons like anthracene and fluorine [Reddy, M. S., Naresh, B., Leela, T., Prashanthi, M., Madhusudhan, N. C., Dhanasri, G. and Devi, P. (2010) Biores. Technol. 101, 7980-7983]. Brevibacterium sp. 7G isolated from a crude oil-contaminated soil also produced a glycolipid biosurfactant [Ferhat, S., Mnif, S., Badis, A., Eddouaouda, K., Alouaoui, R., Boucherit, A., Mhiri, N., Moulai-Mostefa, N. and Sayadi, S. (2011) International Biodeterioration and Biodegradation 65, 1182-1188]. There is paucity of information on EPS producers from Brevibacterium sp., except one report on Brevibacillus thermoruber strain 438 producing EPS [Radchenkova, N., Tomova, A. and Kambourova, M. (2011) Biotechnol. Biotechnol. Eq. 25, 77-79]. Recently, two new bacterial strains of Microbacterium sp. BS-2 and Brevibacillus sp. strain BS-207 were identified based on 16S rDNA sequencing. The 16s rDNA sequences have been deposited in GenBank database with accession numbers, HQ116802 and HQ116803, respectively [Kumar, C. G., Mamidyala, S. K., Sujitha, P., Muluka, H. and Akkenapally, S. (2012) Biotechnol. Prog. 28, 1507-1516].
In view of the above facts, there is an urgent need to identify new and potential biosurfactants that exhibit surface tension lowering activity and other biological properties. The present invention fulfils these requirements as it provides two new bacterial strains of Microbacterium sp. strain BS-2 and Brevibacillus sp. strain BS-207 producing polymeric biosurfactants that has antimicrobial, antioxidant, anti-inflammatory and immunomodulating properties and acted as potential immunosuppressive agents.